Project Summary The primer for reverse transcription in HIV-1, human tRNALys3, is selectively packaged into virions along with tRNALys1,2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor that interacts specifically with both tRNALys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNALys depends on the ability of LysRS to bind to tRNALys and the presence of both host cell factors is required for optimal viral infectivity. LysRS is normally part of a dynamic mammalian multi-synthetase complex. In the previous grant period, we made substantial progress towards elucidating the mechanism by which human LysRS is recruited into HIV-1 particles. We reported that HIV-1 infection results in a free pool of phosphorylated LysRS (pS207-LysRS) that is partially re- localized to the nucleus of target cells. Blocking this pathway in HIV-1 producing cells abolished LysRS packaging and resulted in less infectious progeny virions. We also extended earlier work showing that LysRS binds to a tRNA-like element in the primer-binding site (PBS) region of HIV-1 genomic RNA (gRNA), which facilitates tRNA release from LysRS and primer placement onto the PBS. In the next grant period, we will build on these results by examining the overall hypotheses that phosphorylated LysRS facilitates uncharged tRNA primer packaging and that tRNALys3 annealing to the PBS alters gRNA conformation and dynamics, thereby impacting downstream events that are critical for the HIV-1 lifecycle. This work will transform our understanding of the role of two host cell factors in HIV-1 replication. Aim 1 will explore the role of LysRS phosphorylation in packaging an uncharged form of tRNALys3 and validate this isoform of LysRS as a therapeutic target. Aim 2 will establish the effect of tRNA annealing and pS207-LysRS binding on gRNA structure and dynamics and provide new insights into the timing of this key step in the viral lifecyle, which may reveal a novel therapeutic strategy.